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Acta Agron Sin ›› 2015, Vol. 41 ›› Issue (09): 1353-1360.doi: 10.3724/SP.J.1006.2015.01353

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Enzymatic Characteristics of Acetolactate Synthase Mutant S638N in Brassica napus and Its Resistance to ALS Inhibitor Herbicides

HU Mao-Long, PU Hui-Ming*, LONG Wei-Hua, GAO Jian-Qin, QI Cun-Kou, ZHANG Jie-Fu, CHEN Song   

  1. Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences / Key Laboratory of Cotton and Rapeseed (Nanjing), Ministry of Agriculture / Nanjing Sub-center of National Center of Oil Crops Improvement, Nanjing 210014, China
  • Received:2015-01-05 Online:2015-09-12 Published:2015-09-12

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Abstract: Acetolactate synthase (ALS) catalyses the first step in the synthesis of the branched-chain amino acids and is the target of several classes of herbicides. A BnALS1R gene from herbicide-resistant mutant line M9 in B. napus, was previously isolated and demonstrated to be resistant to the imidazolinone (IMI) herbicides. This research was to reveal the differences of enzymatic characteristics and its resistance to ALS inhibitor herbicides between the mutant S638N and the wild-type enzyme. The BnALS1R gene was constructed and expressed in Escherichia coli along with the wild-type. The target recombinant proteins with the predicted molecular weight (74 kD) were successively expressed in Escherichia coli and purified by SDS-PAGE. The enzymatic activity of the purified S638N and wild-type was then measured in enzyme reaction systems under different temperatures and pH values. Results showed that the S638N resembled the wild-type in their enzymatic activity, showing maximum activity at 37°C and pH 7.0, and no significant difference in the Km and Vmax between the S638N and wild-type. The activation of the S638N by cofactors (FAD, Mg2+ and thiamine diphosphate) was examined and generated similar results to that of the wild-type. The mutant enzyme was inactive when one of three cofactors was omitted. However, the S638N was more resistant to IMI herbicides than the wild-type in contrast to Su herbicides that inhibited the S638N as well as the wild-type. Therefore, the S638N has resistance specific to IMI herbicides with unalteration of the enzymatic reaction characteristics.

Key words: Rapeseed (Brassica napus L.), Imidazolinone herbicides, Acetolactate synthase, S638N, Herbicide-resistance

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